Figure 3.

Pofut1^cax contains an IAP insertion. (A) Map of the region flanking exon5 and PCR-amplified DNA fragments. Mutant DNAs did not produce fragment 4/11 (g, h) but all others (c, d, k, l, o, p) as wild type (wt) (a, b, e, f, i, j, m, n). (B) Map of the 3' region of Pofut1, and Southern blot of wt and mutant DNA hybridized with a cDNA probe containing exon5, 6, and a 5' portion of exon7. Restriction sites and fragments (arrows, labelled by Roman numerals in the scheme and blot) are indicated above and below the map. Asterisks indicate mutant-specific fragments. (C) Long range PCR amplifying fragment 4/11 gave a mutant product (c) larger than wt (a, b). (D) Insertion site map, and junction fragments (1 and 2) amplified with mutant (c, d, g, h) but not wt (a, b, e, f) DNA. Co: water, M: 1 kb ladder. (E) Relative Pofut1 mRNA levels in E10.5 wt (n = 6; white boxes) and mutant embryos (n = 12; gray boxes) determined by exon 6–7 and exon 1–2 amplification. Boxed areas contain 50% of all values. Stippled lines indicate Median expression, whiskers of the boxed areas the total range of values. (F) POFUT1 protein (arrowhead) detected in E9.5 wt embryo extracts was reduced in cax mutants, and absent from Pofut1^{tm1Pst/tm1Pst}. Due to the lower amount of protein in a single growth retarded Pofut1^{tm1Pst/tm1Pst} embryo this lane shows a longer exposure using the non-specific 55 kDa band as an adjustment. Bars indicate molecular weight markers (kDa).